CN115746163A - Sulfated beta-glucan, preparation method and application thereof in immunoprotection and antitumor drugs - Google Patents
Sulfated beta-glucan, preparation method and application thereof in immunoprotection and antitumor drugs Download PDFInfo
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Abstract
The invention belongs to the technical field of chemical synthesis medicines, and particularly relates to sulfated beta-glucan, a preparation method and application thereof in immune protection and antitumor drugs. The invention obtains the sulfated beta-glucan with high purity and good water solubility by sulfating and deriving the insoluble beta-glucan and separating and purifying the insoluble beta-glucan by anion exchange resin. The preparation method is mild and safe, the obtained sulfated beta-glucan has good water solubility and uniform structure, the sulfated beta-glucan can reduce the tumor growth effect by generating immune pre-protection function on a mouse through administration in advance, and can generate good inhibition effect on the tumor growth and tumor metastasis of the mouse under a tumor-bearing model in which B16F10 cells are inoculated subcutaneously and a lung metastasis model in which the cells are injected through tail veins, so that the sulfated beta-glucan can be used for preparing an immunoregulation medicament or a health-care product for resisting tumor growth and tumor metastasis and improving immunity.
Description
Technical Field
The invention belongs to the technical field of chemical synthesis medicines, and particularly relates to sulfated beta-glucan, a preparation method and application thereof in immune protection and antitumor drugs.
Background
Beta-glucan is a kind of polysaccharide composed of glucose residues through beta-glycosidic bonds in different connection modes, beta-glucan compounds are widely existed in nature, natural beta-glucan mainly exists in various eumycetes, bacteria and seaweeds, and beta-glucan with different structures has different biological activities. The structural characteristics are that the main chain is beta-1, 3 glucan, and the beta-1, 3/1, 6-glucan with 6-branch is widely reported to play an anti-tumor role by activating macrophages, natural killer cells and the like and play an anti-inflammatory and antiviral role by enhancing the immune activity. Beta-glucan derived from yeast, schizophyllum commune, euglena and the like is insoluble in water because of the large molecular weight and the formation of a triple helix structure due to the interaction of polyhydroxy, and is called granular beta-glucan, so that the development and application of beta-glucan medicaments are greatly limited. Therefore, it is very important to find a modification method for effectively improving the water solubility of the beta-glucan.
Traditionally, the water solubility of the beta-glucan is improved by obtaining the beta-glucan with low molecular weight through degradation and enzymolysis, but the biological activity is lost at the same time. The water solubility can be improved by carrying out chemical derivative modification on insoluble beta-glucan, so that the beta-glucan derivative with better water solubility can be obtained, and different biological functions can be endowed to the beta-glucan derivative. Chinese patent CN104788581A discloses a sulfation method for improving the water solubility of yeast beta-D-glucan, but they use H 2 SO 4 The sulfuric acid is required to be added dropwise in an ice bath for a sulfating reagent, and the experimental operation has certain dangerousness. Therefore, we further develop a safe and effective sulfation derivation method to obtain water-soluble sulfated beta-glucan, and the application potential of the water-soluble sulfated beta-glucan in immunoprotection and antitumor drugs can be verified through experiments.
The technical problems in the prior art are as follows: at present, no prior art has developed a precedent for preparing sulfated beta-glucan by a safe sulfation derivation method and the application of the sulfated beta-glucan in immunoprotection and antitumor drugs.
The significance of solving the problems and the defects is as follows: (1) The sulfated beta-glucan is prepared by a safe sulfation derivation method for the first time, and the obtained sulfated beta-glucan has good water solubility and uniform structure and can be industrialized on a large scale. (2) The sulfated beta-glucan disclosed by the invention is used for designing a pre-protection model by a principle of 'first administration and then inoculation', and designing a therapeutic model by a principle of 'first inoculation and then administration', and the sulfated beta-glucan has an immune protection effect by evaluation of two mouse models. (3) The sulfated beta-glucan disclosed by the invention can be used for creating a tumor-bearing model for mouse melanoma high-metastasis cells B16F10 in a subcutaneous tumor mode, and has a remarkable inhibiting effect on the growth of the tumor volume. The sulfated beta-glucan disclosed by the invention is used for making a lung metastasis model by a tail vein injection mode on mouse melanoma high-metastasis cells B16F10, and has a remarkable inhibiting effect on the number of black nodules transferred to mouse lung tissues.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides sulfated beta-glucan, a preparation method and application thereof in immune protection and antitumor drugs.
In order to achieve the purpose, the invention adopts the following technical scheme:
a sulfated beta-glucan having the structure:
further, hydroxyl (OH) on the glucose ring in the beta-glucan is substituted by sulfate (OSO) 3 Na) is substituted, the substitution degree is that 1 to 3 hydroxyl groups on each sugar monomer are substituted by sulfate groups, and the total mass ratio of the sulfate groups is 0.01 to 30 percent.
A method for preparing sulfated beta-glucan, wherein the sulfated beta-glucan is obtained by substituting hydroxyl on a sugar ring with a sulfate group through a chemical modification method, and the method comprises the following steps:
step 1: dissolving beta-glucan and a sulfating reagent in N, N-Dimethylformamide (DMF), and stirring and reacting for 12-72 hours under 40-90 ℃ oil bath;
step 2: after the reaction is finished, adjusting the pH of the reaction solution to be =8 by 0.2-2M sodium hydroxide, decompressing and concentrating to 1/10 of the volume of the original solution, adding 4-32% of sodium chloride solution with the same volume, mixing uniformly, adding 4-5 times of ethanol, mixing uniformly, centrifuging for 10 minutes at 8000r/min, adding 4-32% of sodium chloride solution into the precipitate until the sodium chloride solution is just dissolved, continuing alcohol precipitation, repeating for 3 times, collecting the precipitate, dialyzing for 48 hours by a 3000Da dialysis bag, finishing dialysis, decompressing and concentrating the solution, and freeze-drying to obtain the crude product of the unpurified sulfated beta-glucan.
And 3, step 3: the step of purifying the unpurified crude sulfated beta-glucan comprises the steps of dissolving the unpurified crude sulfated beta-glucan obtained by the previous step with distilled water, taking an aqueous sodium chloride solution as a mobile phase, separating and purifying through anion exchange resin, detecting through a sulfuric acid phenol method, merging and collecting components containing polysaccharide, dialyzing through distilled water, concentrating under reduced pressure, and freeze-drying to obtain the high-purity sulfated beta-glucan.
Preferably, the molecular weight of the beta-glucan is 3-1500 kDa, the monosaccharide mainly comprises glucose (Glc), the main chain is formed by connecting glucose through beta-1, 3 glycosidic bonds, and the main chain glucose is provided with beta-1, 6 branches, and the branching degree is 1-60%.
Preferably, the sulfating agent used in step 1 is sulfur trioxide triethylamine (SO) 3 ·Et 3 N), sulfur trioxide pyridine (SO) 3 Py) or chlorosulfonic acid pyridine (ClSO) 3 H Py) and mixtures thereof.
Preferably, the crude sulfated β -glucan is purified using an aqueous sodium chloride solution having a molar concentration of 0.1 to 2.5M, and the anion exchange resin is at least one of diethylaminoethyl cellulose (DEAE-cellulose), DEAE-dextran (DEAE-Sephadex), DEAE-agarose (DEAE-Sepharose), and Q-Sepharose (Q-Sepharose).
The sulfated beta-glucan is used in anti-tumor growth and anti-tumor metastasis medicaments, immunity-improving immunoregulation medicaments or health care products.
Further, the application of the sulfated beta-glucan in the aspect of anti-tumor metastasis drugs is specifically shown as follows: a mouse tumor-bearing model is made by inoculating a mouse melanoma high-metastasis cell B16F10 to a mouse subcutaneously, and the tumor size of the mouse tumor-bearing model is remarkably inhibited; promoting the secretion of cell factors such as TNF-alpha, IL-beta and the like in serum of a tumor-bearing model mouse, and further playing a role in inhibiting the growth of tumors; the mouse melanoma high-metastasis cell B16F10 is used for making a lung metastasis model by a tail vein injection mode for a mouse, and has a remarkable inhibiting effect on the number of black knots transferred to mouse tissues; promote the secretion of cell factors such as TNF-alpha, IL-beta and the like in serum of a metastatic model mouse, and further play a role in inhibiting tumor metastasis; has obvious binding capacity with fibroblast growth factor FGF, vascular endothelial growth factor VEGF and the like; the medicine is in the form of injection, powder injection, oral liquid, tablet, capsule, soft capsule and powder.
Further, the application of the sulfated beta-glucan in the immunoregulation medicaments or health-care products for improving the immunity is specifically shown as follows: the pre-protection model is designed by the principle of 'first administration and then inoculation', the therapeutic model is designed by the principle of 'first inoculation and then administration', and the sulfated beta-glucan has immune protection effect which can be evaluated by two mouse models; the medicine or health product is in the form of injection, powder injection, oral liquid, tablet, capsule, soft capsule and powder.
To verify the technical effect of sulfated β -glucans, the inventors made the following experiments:
the technical effect is as follows: the use of sulfated beta-glucan for immune modulation to improve immunity, in designing a pre-protective model by the "administration first followed by vaccination" principle, and in designing a therapeutic model by the "vaccination first followed by administration" principle. By comparing the tumor volumes of model mice for both dosing regimens, the results indicate that sulfated β -glucan has immunoprotective effects. Specifically, the sulfated beta-glucan enables a mouse to have a stronger immune protection effect in a pre-administration scheme, and the tumor volume of the sulfated beta-glucan is obviously reduced compared with that of a model group; the tumor volume of the mice is also reduced in the post-dosing regimen compared with the model group, but the effect is weaker than that of the pre-dosing regimen, and the result shows that the sulfated beta-glucan has an immunoprotective effect on tumor model mice, and the tumor inhibition effect of the sulfated beta-glucan is remarkable in the pre-dosing regimen.
The experimental verification steps are as follows:
(a) The concrete modeling mode of the pre-protection model mouse is as follows: after 7-week-old C57 mice were acclimatized for 2 weeks, the sulfated beta-glucan was administered to the mice at a dose of 4mg/kg once every 3 days by tail vein administration. After the 7 th dose, after culturing a certain number of B16F10 cells and resuspending them with PBS, the cell count was 5-45X 10 5 mL, subcutaneously inoculated in the right axilla of C57 mice at a dose of 100 microliters per mouse. Monitoring the growth of tumor volume every day until the tumor volume reaches 1500mm 3 The experiment was stopped.
(b) The concrete modeling mode of the therapeutic model mouse is as follows: 7 weeks old C57 mice, after acclimatization for 2 weeks, cultured a certain number of B16F10 cells, and PBS heavy suspension, cell count 5 ~ 45X 10 5 mL, subcutaneously inoculated in the right axilla of C57 mice at a dose of 100 microliters per mouse. Monitoring the growth condition of the tumor volume every day, and waiting for the tumor volume to grow to 100mm in 5-10 days 3 At this time, the mice were administered the sulfated β -glucan at a dose of 4mg/kg once every 3 days by tail vein administration. Monitoring the growth of the tumor volume every day until the tumor volume reaches 1500mm 3 The experiment was stopped.
The mice used were C57BL/6J Nifdc male and female mice; the age of the mouse is 6-8 weeks; 5-45X 10 for each C57 mouse 4 (ii) individual cell dose inoculation; tumor volume was calculated as V =0.5 × (L × W) 2 ) Wherein L is the tumor length (mm) and W is the tumor width (mm).
The technical effect is that the sulfated beta-glucan has the functions of resisting tumor growth and tumor metastasis.
(1) A mouse tumor-bearing model is made by using mouse melanoma high-metastasis cells B16F10 to a mouse through a subcutaneous inoculation mode, and the mouse melanoma high-metastasis cells have a remarkable inhibition effect on the size of a tumor. The mice were sacrificed and tumor tissues were weighed and photographed, and the results showed that tumor volume and weight could be significantly reduced by low dose (4 mg/kg) sulfated β -glucan (SCGS-4) in the pre-dosing regimen, and that the tumor suppression effect was more significant than that of the non-pre-dosed group (SCGS-UT 8) by pre-dosing (SCGS-8) before tumor inoculation at the same dosing concentration.
The specific molding method comprises the following steps: after a certain number of cultured B16F10 cells are resuspended in PBS, the cell count is 5-45 × 10 5 mL, subcutaneously inoculated in the right axilla of C57 mice at a dose of 100 microliters per mouse. The tumor volume can be reached and measured after 5-10 days and is 100mm 3 。
The secretion of cytokines such as TNF-alpha and IL-beta and the like in serum of a tumor-bearing model mouse is detected by an Elisa kit, sulfated beta-glucan (SCGS-8) can also obviously improve the secretion of the cytokines of the innate immune system, and the effect is more obvious than that of a non-predose group (SCGS-UT 8) under the same administration concentration, which indicates that the sulfated beta-glucan can improve the immune protection effect of the model mouse by pre-administration and further play a role in inhibiting tumor growth.
(2) A lung metastasis model is made by injecting mouse melanoma high-metastasis cells B16F10 into a mouse through tail vein, and the lung metastasis model has a remarkable inhibiting effect on the number of black knots transferred to mouse tissues. The result of taking lung tissues after the mice are sacrificed to show that black nodules are counted and photographed shows that the lung tissues can be obviously reduced to show that the black nodules are obtained by low dose (4 mg/kg) of sulfated beta-glucan (SCGS-4) in a pre-dosing scheme, and the effect is more obvious than that of a positive control group (LMWH).
The specific molding method comprises the following steps: after a certain number of cultured B16F10 cells were resuspended in PBS, the cell count was 5-45X 10 5 mL, 100 μ l per mouse were inoculated into the tail vein of C57 mice by tail vein injection.
The secretion of cell factors such as TNF-alpha, IL-beta and the like in serum of a metastatic model mouse is detected by an Elisa kit, and the result shows that sulfated beta-glucan (SCGS-8) can also obviously improve the secretion of cell factors of an innate immune system, the effect is more obvious than that of a positive control group (LMWH), and the sulfated beta-glucan can improve the immune protection effect of the model mouse through pre-administration, so that the tumor metastasis inhibition effect is further exerted.
The experiment finds that: has obvious binding capacity with fibroblast growth factor FGF, vascular endothelial growth factor VEGF and the like, and is evaluated by a Surface Plasmon Resonance (SPR) technology, and K combined with FGF1 D K binding to FGF2 =2.339 nM D K binding to VEGF of =0.996nM D ＝3.728nM。
The invention has the beneficial technical effects that: (1) The preparation method of sulfated beta-glucan has the advantages of mild and safe conditions, no use of sulfuric acid, good water solubility of the obtained sulfated beta-glucan, uniform structure and industrialization suitability. (2) The sulfated beta-glucan disclosed by the invention is used for designing a pre-protection model by a principle of 'first administration and then inoculation', and designing a therapeutic model by a principle of 'first inoculation and then administration', and two mouse models can evaluate that the sulfated beta-glucan has an immunoprotection effect and provides a basis for the sulfated beta-glucan in immunopotentiators, health care products and medicines for developing and inhibiting tumor growth. (3) The sulfated beta-glucan disclosed by the invention can be used for constructing a tumor-bearing model for mouse melanoma high-metastasis cells B16F10 in a subcutaneous tumor connection mode, and has a remarkable inhibiting effect on the growth of the tumor volume. The sulfated beta-glucan disclosed by the invention is used for making a lung metastasis model by a tail vein injection mode on mouse melanoma high-metastasis cells B16F10, and has a remarkable inhibiting effect on the number of black nodules transferred to mouse lung tissues. Provides a basis for the development of sulfated beta-glucan as a medicament for inhibiting tumor growth and metastasis.
Drawings
FIG. 1 route to sulfated beta-glucan preparation;
FIG. 2 DEAE Sephadex elution profile of sulfated beta-glucan;
FIG. 3 molecular weight distribution plot of sulfated β -glucan;
FIG. 4 is a comparison graph of nuclear magnetic hydrogen spectra before and after sulfation modification of beta-glucan;
FIG. 5 sulfated beta-glucan immunoprotection;
FIG. 6 sulfated beta-glucan inhibits tumor growth;
FIG. 7 sulfated beta-glucan inhibits tumor metastasis;
examples
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
Example 1: preparation of sulfated beta-glucans
The sulfated beta-glucan is obtained by substituting hydroxyl on a sugar ring with a sulfate group by a chemical modification method of the beta-glucan, the reaction route is shown as figure 1, and the preparation of the sulfated beta-glucan specifically comprises the following steps:
step 1): 50mg of beta-glucan and 750mg of sulfating agent (SO) 3 ·Et 3 N) was dissolved in 4mL of DMF, and the reaction was stirred in a 70 ℃ oil bath for 24 hours.
Step 2): after the reaction, 1M sodium hydroxide solution was slowly added dropwise to adjust the reaction solution to pH =8, and the solution was concentrated under reduced pressure to 1/10 of the volume of the original solution. Adding 16% sodium chloride solution with the same volume as the volume of the mixture, mixing the mixture evenly, adding ethanol with the volume of 4-5 times of the volume of the mixture, mixing the mixture evenly, centrifuging the mixture for 10 minutes at 8000r/min, adding a proper amount of 16% sodium chloride solution into the precipitate for dissolution, continuing alcohol precipitation, repeating the process for 3 times, collecting the precipitate, and dialyzing the precipitate for 48 hours by a 3000Da dialysis bag. After dialysis, the solution is decompressed, concentrated and freeze-dried to obtain the unpurified crude product of sulfated beta-glucan.
Step 3): the method for refining the unpurified crude sulfated beta-glucan comprises the following steps: dissolving the obtained unpurified sulfated beta-glucan crude product by using distilled water, respectively taking 0M, 0.5M, 1M, 1.5M and 2M sodium chloride aqueous solutions as mobile phases, respectively collecting 3 column volumes through anion exchange resin DEAE-Sephadex Fast Flow, detecting the polysaccharide content by a sulfuric acid phenol method, collecting 1.5M sodium chloride elution components, dialyzing by using distilled water through a 3000Da dialysis bag for 48 hours, and freeze-drying after the solution is subjected to reduced pressure concentration to obtain the high-purity sulfated beta-glucan.
Example 2: preparation of sulfated beta-glucans
Step 1): 50mg of beta-glucan and 500mg of the sulfating agent sulfur trioxide triethylamine (SO) 3 ·Et 3 N) was dissolved in 4mL of DMF and the reaction was stirred in an oil bath at 90 ℃ for 24 hours.
Step 2): after the reaction, 1M sodium hydroxide solution was slowly added dropwise to adjust the reaction solution to pH =8, and the solution was concentrated under reduced pressure to 1/10 of the volume of the original solution. Adding an isovolumetric 32% sodium chloride solution, mixing uniformly, adding ethanol with the volume of 4-5 times, mixing uniformly, centrifuging for 10 minutes at 8000r/min, adding 32% sodium chloride solution into the precipitate until the precipitate is just dissolved, continuing alcohol precipitation, repeating for 3 times, collecting the precipitate, and dialyzing for 48 hours by a 3000Da dialysis bag. After the dialysis, the solution is decompressed and concentrated and then is frozen and dried to obtain the unpurified crude product of the sulfated beta-glucan.
Step 3): the method for refining the unpurified crude product of sulfated beta-glucan comprises the following steps: dissolving the obtained unpurified sulfated beta-glucan crude product by using distilled water, respectively taking 0M, 0.5M, 1M, 1.5M and 2M sodium chloride aqueous solutions as mobile phases, respectively collecting 3 column volumes by anion exchange resin DEAE-Sephadex Fast Flow, detecting the content of polysaccharide by a sulfuric acid phenol method, collecting 1.5M sodium chloride elution components, dialyzing by using distilled water through a 3000Da dialysis bag for 48 hours, and freeze-drying after the solution is subjected to reduced pressure concentration to obtain the high-purity sulfated beta-glucan.
Example 3: preparation of sulfated beta-glucans
Step 1): 50mg of beta-glucan and 300mg of the sulfating agent sulfur trioxide triethylamine (SO) 3 ·Et 3 N) was dissolved in 4mL DMF. The reaction was stirred for 24 hours at 40 ℃ in an oil bath.
Step 2): after the reaction, 1M sodium hydroxide solution was slowly added dropwise to adjust the reaction solution to pH =10, and the solution was concentrated under reduced pressure to 1/10 of the volume of the original solution. Adding an isovolumetric 32% sodium chloride solution, mixing uniformly, adding ethanol with the volume of 4-5 times, mixing uniformly, centrifuging for 10 minutes at 8000r/min, adding 32% sodium chloride solution into the precipitate until the precipitate is just dissolved, continuing alcohol precipitation, repeating for 3 times, collecting the precipitate, and dialyzing for 48 hours by a 3000Da dialysis bag. After the dialysis, the solution is decompressed and concentrated and then is frozen and dried to obtain the unpurified crude product of the sulfated beta-glucan.
And step 3): the method for refining the unpurified crude product of sulfated beta-glucan comprises the following steps: dissolving the obtained unpurified sulfated beta-glucan crude product by using distilled water, respectively taking 0M, 0.5M, 1M, 1.5M and 2M sodium chloride aqueous solutions as mobile phases, respectively collecting 3 column volumes by anion exchange resin DEAE-Sephadex Fast Flow, detecting the content of polysaccharide by a sulfuric acid phenol method, collecting 1.5M sodium chloride elution components, dialyzing by using distilled water through a 3000Da dialysis bag for 48 hours, and freeze-drying after the solution is subjected to reduced pressure concentration to obtain the high-purity sulfated beta-glucan.
Example 4: structural characterization of sulfated beta-glucans
The structural characterization of the polysulfated beta-glucan of the invention specifically comprises the following steps:
step 1): absolute molecular weight determination and purity analysis: the absolute molecular mass of the sulfated beta-glucan was determined and the purity was analyzed by High Performance Gel Permeation Chromatography (HPGPC) -eighteen Angle Laser Light Scattering (MALLs) in combination.
Chromatographic conditions are as follows: a chromatographic column: shodex OHPPak SB 803HQ chromatographic column is connected with Shodex OHPPak SB804 HQ chromatographic column in series; mobile phase: 0.1mol of L-1Na 2 SO 4 An aqueous solution; the differential detector and the eighteen-angle laser light scattering instrument are connected in series to detect and measure the molecular weight of the polyglucuronic acid on line. As shown in fig. 3, the sulfated β -glucan sample had a uniform peak shape at 17 to 25 minutes in the spectrum, and the sample was highly pure and had an absolute molecular mass of 146kDa as measured.
Step 2): nuclear magnetic resonance spectrogram analysis: taking 10mg sulfated beta-glucan, adding 500 mu L of heavy water, freeze-drying, repeatedly performing 3 times of heavy water exchange, adding 500 mu L of heavy water and transferring to a nuclear magnetic tube, and performing nuclear magnetic resonance spectrum analysis at 25 ℃ by taking deuterated acetone as an internal standard. The results are shown in fig. 4, where the nuclear magnetic hydrogen spectrum of β -glucan shifts overall to a low field after sulfated derivatization, indicating that sulfated derivatization is complete.
Example 5: immunoprotection assay of sulfated beta-glucan
The pre-protection model is designed by the principle of 'first administration and then inoculation', the therapeutic model is designed by the principle of 'first inoculation and then administration', and the sulfated beta-glucan has immune protection effect which can be evaluated by two mouse models. The results are shown in fig. 5, where sulfated β -glucan in a multiple pre-dosing regimen provided a strong immunoprotection in mice with significantly reduced tumor volume compared to the model group, by comparing the tumor volume of the model mice of the two dosing regimens; the tumor volume of the mice is also reduced in the post-dosing scheme compared with the tumor volume of the model group, but the effect is weaker than that of the pre-dosing scheme, and the result shows that the sulfated beta-glucan has an immune protection effect on the tumor model mice, and the tumor inhibition effect of the sulfated beta-glucan is obvious in the pre-dosing scheme.
Step 1): the specific modeling mode of the pre-protection model mouse is as follows: 7 weeks old C57BL/6J Nifdc male mice, after acclimatization and 2 weeks, mice were administered with the sulfated beta-glucan as described above at a dose of 4mg/kg once every 3 days by tail vein administration. After the 7 th dose, after culturing a certain number of B16F10 cells and resuspending them with PBS, the cell count was 5-45X 10 5 mL, subcutaneously inoculated in the right axilla of C57 mice at a dose of 100 microliters per mouse. Monitoring the growth of the tumor volume every day until the tumor volume reaches 1500mm 3 The experiment was stopped.
Step 2) the therapeutic model mouse is specifically modeled as follows: c57BL/6J Nifdc male mice of 7 weeks old are adapted to the environment for 2 weeks, cultured a certain number of B16F10 cells are resuspended by PBS, and the cell count is 5-45 multiplied by 10 5 mL, subcutaneously inoculated in the right axilla of C57 mice at a dose of 100 microliters per mouse. Monitoring the growth of tumor volume every day, and waiting for the tumor volume to grow to 100mm in 5-10 days 3 At first, mice were administered the sulfated β -glucan described above at a dose of 4mg/kg once every 3 days by tail vein administration. Every day supervisionMeasuring the growth of the tumor volume until the tumor volume reaches 1500mm 3 The experiment was stopped.
Example 6: sulfated beta-glucan test for Secondary antitumor growth
A tumor-bearing model is constructed by utilizing a mode of subcutaneous tumor grafting of mouse melanoma high-metastatic cells B16F10, and the sulfated beta-glucan is found to have the effect of resisting tumor growth, and is shown to be capable of obviously reducing the tumor volume and weight of a model mouse and promoting the secretion of a cytokine of an innate immune system.
The concrete molding method of the tumor-bearing model comprises the following steps: mice were administered the sulfated ss-glucan described above via the tail vein at a low dose of 4mg/kg, a medium dose of 8mg/kg, and a high dose of 16mg/kg, respectively, at doses administered once every 3 days. After the 7 th administration, after culturing a certain number of B16F10 cells and resuspending them with PBS, the cell count was 5 to 45X 10 5 mL, subcutaneously inoculated in the right axilla of C57 mice at a dose of 100 microliters per mouse. The tumor volume can be reached and measured after 5-10 days and is 100mm 3 . During the period, the medicine is continuously administrated according to the set dose, the growth condition of the tumor volume is monitored every day, and the tumor volume is kept to grow to 1500mm 3 The experiment was stopped.
The mice were sacrificed and tumor tissues were taken and weighed and photographed, and the results are shown in fig. 6, the tumor volume and weight were significantly reduced by the low dose (4 mg/kg) of sulfated beta-glucan (SCGS-4) in the pre-dosing regimen, and the tumor-inhibiting effect was more significant than that of the non-pre-dosed group (SCGS-UT 8) by pre-dosing (SCGS-8) before tumor inoculation at the same dosing concentration; the secretion of cytokines such as TNF-alpha, IL-1 beta and the like in serum of a tumor-bearing model mouse is detected by an Elisa kit, sulfated beta-glucan (SCGS-8) can also obviously improve the secretion of the cytokines of an innate immune system, and the effect is more obvious than that of a non-predose group (SCGS-UT 8) under the same administration concentration, which indicates that the sulfated beta-glucan can improve the immune protection effect of the model mouse through predose and further play a role in inhibiting the growth of tumors.
Example 7: experiment of anti-tumor metastasis effect of sulfated beta-glucan
A lung transfer model is made by injecting mouse melanoma high-transfer cells B16F10 into a mouse through tail vein, and sulfated beta-glucan is found to have the function of resisting tumor transfer and is shown to be capable of obviously reducing the number of black knots shown by model mouse tissues.
The specific molding method comprises the following steps: mice were administered the sulfated ss-glucan described above via the tail vein at a dose of 4mg/kg at the low dose, 8mg/kg at the medium dose, and 16mg/kg at the high dose, respectively, and once every 3 days. After the 7 th dose, after culturing a certain number of B16F10 cells and resuspending them with PBS, the cell count was 5-45X 10 5 mL, 100 microliters per mouse was inoculated into the tail vein of C57 mice by tail vein injection. The dosing was continued at the set dose during which the experiment was stopped 15 days after tumor cell inoculation.
The secretion of cytokines such as TNF-alpha, IL-beta and the like in serum of a transfer model mouse is detected by an Elisa kit, so that the effect of inhibiting tumor transfer is exerted.
After the mice are sacrificed, lung tissues are taken to indicate black nodules and are counted and photographed, the result is shown in figure 7, the lung tissues can be obviously reduced by low dose (4 mg/kg) of sulfated beta-glucan (SCGS-4) in a pre-administration scheme to indicate the black nodules, and the effect is more obvious than that of a positive control group (LMWH); the secretion of cell factors such as TNF-alpha, IL-1 beta and the like in serum of a tumor-bearing model mouse is detected by an Elisa kit, sulfated beta-glucan (SCGS-8) can also obviously improve the secretion of the cell factors of an innate immune system, and the effect is more obvious than that of a positive control group (LMWH), which indicates that the sulfated beta-glucan can improve the immune protection effect of the model mouse by pre-administration, and further plays a role in inhibiting tumor metastasis.
In conclusion, the sulfated beta-glucan is derived from the beta-glucan serving as a raw material through chemical modification, wherein the molecular weight of the beta-glucan is 3 kDa-1500 kDa, the monosaccharide composition is mainly glucose (Glc), the connection mode of the beta-glucan is a main chain beta-1, 3 glucan structure, the main chain glucose has beta-1, 6 branches, and the branching degree is 1% -60%; by reaction of sulfur trioxide triethylamine (SO) 3 ·Et 3 N) and sulfur trioxide pyridine (SO) 3 Py) in beta-glucan by substituting the hydroxyl groups of the glucose ring with sulfate groups, with the degree of substitution being per saccharide monomer1 to 4 hydroxyl groups are substituted by sulfate radicals, and the total mass ratio of the sulfate radicals is 0.01 to 30 percent. The sulfated beta-glucan has the functions of generating immune pre-protection function on mice through early administration so as to reduce the tumor volume growth, and can obviously inhibit the tumor growth and tumor metastasis inhibition of B16F10 cells in a tumor-bearing model inoculated subcutaneously and a lung metastasis model mouse injected through tail vein. Can be used for preparing antitumor drugs, immunopotentiators, health products and special medical application formula foods.
The above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that the technical solutions described in the foregoing embodiments may be modified or part of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A sulfated β -glucan, wherein the sulfated β -glucan has a structural formula:
2. the sulfated beta-glucan according to claim 1, wherein: the hydroxyl (OH) on the glucose ring of the beta-glucan is replaced by sulfate (OSO) 3 Na) is substituted, the substitution degree is that 1 to 4 hydroxyl groups on each sugar monomer are substituted by sulfate groups, and the total mass ratio of the sulfate groups is 0.01 to 30 percent.
3. A method of producing a sulfated β -glucan as claimed in claim 1, wherein:
the sulfated beta-glucan is obtained by substituting hydroxyl on a sugar ring with sulfate by a chemical modification method through the beta-glucan, and the preparation method comprises the following steps:
step 1: dissolving beta-glucan and a sulfating reagent in N, N-Dimethylformamide (DMF), and stirring and reacting for 12-72 hours under 40-90 ℃ oil bath;
and 2, step: after the reaction is finished, adjusting the pH of the reaction solution to be =8 by 0.2-2M sodium hydroxide, concentrating under reduced pressure to 1/10 of the volume of the original solution, adding 4-32% of sodium chloride solution with the same volume, mixing uniformly, adding 4-5 times of ethanol, mixing uniformly, centrifuging at 8000r/min for 10 minutes, adding 4-32% of sodium chloride solution into the precipitate until the sodium chloride solution is just dissolved, continuing alcohol precipitation, repeating for 3 times, collecting the precipitate, dialyzing for 48 hours by a 3000Da dialysis bag, after the dialysis is finished, concentrating the solution under reduced pressure, and freeze-drying to obtain an unpurified crude product of sulfated beta-glucan.
And step 3: the method comprises the following steps of purifying an unpurified crude sulfated beta-glucan, dissolving the obtained unpurified crude sulfated beta-glucan with distilled water, taking a sodium chloride aqueous solution as a mobile phase, separating and purifying through anion exchange resin, detecting through a sulfuric acid phenol method, combining and collecting components containing polysaccharide, dialyzing through distilled water, concentrating under reduced pressure, and freeze-drying to obtain the high-purity sulfated beta-glucan.
4. The method of producing a sulfated β -glucan as claimed in claim 3, wherein: the molecular weight of the beta-glucan is 3-1500 kDa, the monosaccharide composition is mainly glucose (Glc), the main chain is formed by connecting glucose through beta-1, 3 glycosidic bonds, and meanwhile, the glucose on the main chain is provided with beta-1, 6 branches, and the branching degree is 1-60%.
5. The method of producing a sulfated β -glucan as claimed in claim 3, wherein: the sulfating reagent used in the step 1 is sulfur trioxide triethylamine (SO) 3 ·Et 3 N), sulfur trioxide pyridine (SO) 3 Py) or chlorosulfonic acid pyridine (ClSO) 3 H Py) and mixtures thereof.
6. The method of producing a sulfated β -glucan as claimed in claim 3, wherein: the molar concentration of the aqueous sodium chloride solution used for refining the crude sulfated beta-glucan without purification is 0.1 to 2.5M, and the anion exchange resin is at least one of diethylaminoethyl cellulose (DEAE-cellulose), DEAE-dextran (DEAE-Sephadex), DEAE-agarose (DEAE-Sepharose) and Q-Sepharose (Q-Sepharose).
7. Use of a sulfated beta-glucan as claimed in any one of claims 1 to 7 in the preparation of a medicament for the treatment of tumor growth and tumor metastasis.
8. Use of a sulfated beta-glucan as claimed in any one of claims 1 to 7 in the preparation of an immunomodulatory drug or health product for enhancing immunity.
9. Use according to claim 7, characterized in that: the application of the sulfated beta-glucan in preparing the medicaments for resisting tumor growth and tumor metastasis is specifically shown as follows: a mouse tumor-bearing model is made by inoculating a mouse melanoma high-metastasis cell B16F10 to a mouse subcutaneously, and the tumor size of the mouse tumor-bearing model is remarkably inhibited; promoting the secretion of cell factors such as TNF-alpha, IL-beta and the like in serum of a tumor-bearing model mouse, and further playing a role in inhibiting the growth of tumors; the mouse melanoma high-metastasis cell B16F10 is used for making a lung metastasis model by a tail vein injection mode for a mouse, and has a remarkable inhibiting effect on the number of black knots transferred to mouse tissues; promoting the secretion of cytokines such as TNF-alpha, IL-beta and the like in serum of a transfer model mouse, and further playing a role in inhibiting tumor transfer; has obvious binding capacity with fibroblast growth factor FGF and vascular endothelial growth factor VEGF; the medicine is in the form of injection, powder injection, oral liquid, tablet, capsule, soft capsule and powder.
10. Use according to claim 8, characterized in that: the application of the medicine or health-care product for improving immunity is characterized in that a pre-protection model is designed by the principle of 'first administration and then inoculation', a therapeutic model is designed by the principle of 'first inoculation and then administration', and sulfated beta-glucan has an immunoprotection effect through evaluation of two mouse models, wherein the medicine or health-care product is in the forms of injection, powder injection, oral liquid, tablets, capsules, soft capsules and powder.
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